Prolonged and high consumption of alcohol (ethanol) may lead to serious alcohol-related diseases and alcoholism in man. On the other hand, a low to moderate consumption is commonly accepted as being associated with a reduced risk of coronary heart disease, possibly due to an antiatherogenic effect of ethanol. In order to be able to classify a person's drinking behavior, or to more accurately direct medical investigations in the search for an underlying cause of one or several observed symptoms, slow eliminating bio-markers of ethanol are highly useful as objective measures of the person's historic alcohol consumption. Phosphatidylethanol (PEth), an unnatural phospholipid formed by phospholipase D catalyzed ethanolysis in-vivo of mainly endogenous phosphatidylcholine, has recently gained high interest as such a slow eliminating bio-marker. Today, LC-MS-based analytical techniques are commonly used for the quantification of PEth in human blood-samples. One or a few PEth-homologues, such as PEth-16:0/18:1 and/or PEth-16:0/18:2, are usually quantified and used as a representative measure of the total PEth-level.
Nalesso et al. (Journal of Chromatography, 2011, 8423-8431) describe the development of a novel LC-HRMS based method for the quantitative profiling of PEth molecular species in human blood from heavy and social drinkers. Commercially available chloroform solutions of the sodium salt of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanol (PEth-16:0/16:0), 1,2-dioleoyl-sn-glycero-3-phosphoethanol (PEth-18:1/18:1) and 1,2-dioleoyl-sn-glycero-3-phosphobutanol (PBut-18:1/18:1) were used as reference materials and internal standard, respectively, for the qualitative and/or quantitative determination of various PEth-homologues.
Zheng et al. (Clinica Chimica Acta, 2011, 1428-1435) describe an LC-ESI-MS(/MS) method for the simultaneous qualitative and quantitative determination of PEth forms in whole blood samples. Commercially available or synthetically produced PEth-16:0/18:1, PEth-16:0/18:2, deuterium labeled PEth-16:0/18:1, deuterium labeled PEth-16:0/18:2 and phosphatidylpropanol (PProp-18:1/18:1) were used as reference materials or internal standards for the qualitative and/or quantitative determination of various PEth-homologues.
WO2012005680 A1 discloses methods for assessment of previous ethanol exposure by obtaining a ratio between one or several bio-precursors of PEth and one or several PEth-homologues in a sample. Methods comprising the step of removal of the carboxylic acid residues of PEth by selective hydrolysis or selective transesterification followed by quantification of the resulting respective products are also disclosed.
Quantitative nuclear magnetic resonance (QNMR) spectroscopy is widely accepted as a qualitative and quantitative analytical tool. QNMR may be used as an alternative or complementary technique to traditional analytical methods, such as e.g. methods relying on chromatographic separation of organic compounds, for the quantification of one or several compounds of interest. A major advantage of QNMR, in comparison to most traditional analytical methods, is the lack of requirement of an identical standard reference material for quantitative determination of an organic compound of interest in solution. Useful nuclei for quantitative determination employing QNMR-techniques include 1H (1H-QNMR), 31P (31P-QNMR) and 19F (19F-QNMR), of which the former is generally the most useful due a wide occurrence in combination with generally sharp signals, from which desired accurate and precise raw data may be collected.
JPH11174139 A1 discloses a method for quantitative determination of a mixture comprising a surface active agent utilizing 1H-QNMR by employment of an internal standard.
Nuclear magnetic resonance (NMR) analysis of the 1H- (1H-NMR) and 13C- (13C-NMR) nuclei of lipophilic lipids, including PEth, its less lipophilic major bio-precursor phosphatidylcholine (PC) and the structurally related phosphatidylethanolamine (PE), is most commonly conducted in fat solubilizing lipophilic solvents, typically deuterated chloroform (CDCl3), with an optional additive of deuterated methanol (CD3OD) as a minor component in the case of e.g. PC.
Kihara et al. (Chem. Pharm. Bull., 1994, 289-292) used CDCl3 as solvent for the 1H-NMR analysis of PE-16:0/18:2 (compound 12) and the sodium salt of 1-palmitoyl-2-linoleoylphosphatidylmethanol (compound 13), the latter of which is highly structurally and physiochemically similar to PEth-16:0/18:2.
Willmann et al. (Journal of Biomedicine and Biotechnology, 2011, 1-8) used a 2:1-mixture of CDCl3 and deuterated methanol (CD3OD) for the 1H- and 13C-NMR analysis of various phosphatidylcholines (PCs).
Lehnhardt et al. (NMR Biomed. 2001, 307-317) used deuterated water (D2O) as solvent and 3-(trimethylsilyl)propionic acid as internal standard in the quantitative determination by 1H-QNMR of various phospholipids, although not including PEth or any other alkyl-analog thereof. Quantitative determination of such lipids employing 31P-QNMR in a matrix comprising a 8:2-mixture of water and D2O, Na-cholate and EDTA, using phosphono-methyl-glycerol as internal reference, is also described therein.
Vyssotski et al. (Lipids, 2009, 381-389) describes quantitative determination of various lipids, including PEth and alkyl analogs, employing 31P-QNMR in a matrix comprising a 8:2-mixture of water and D2O, Na-cholate and EDTA, using phosphonomethylglycine as internal reference.
Lancée-Hermkens et al. (Biochimica et Biophysica Acta, 1977, 141-151) describes 13C-NMR analysis of PC bilayers of different fatty acid and sterol composition. NMR-spectra were run in a D2O-based matrix. It was observed that cholesterol interacted with PC, which interaction generally manifested itself in an increased line broadening of the 13C-NMR signals of the hydrophobic part of the PC. This result suggests that 1H-NMR signals of similar lipids, e.g. PEth, may broaden in the presence of other amphiphilic components, such as e.g. Na-cholate or the like, in a polar solvent.
Currently, all LC-MS-based methods employed for quantitative determination of phosphatidylalkanols, such as e.g. PEth, in blood samples, including those mentioned herein above, rely on analytical reference solutions, typically solutions comprising a single phosphatidylalkanol or analog in which the concentration thereof is known, for development of standard curves for quantification and/or quantitative instrument calibration. The standard laboratory technique, used for the preparation of such reference solutions involve precise weighing of the reference compound, followed by addition to a precise volume of a suitable solvent. The error in the final concentration of the reference is dependent of factors including e.g. weighing-errors, errors in the assumption of the purity of the reference material, error in the measurement of the volume of solvent and errors related to incomplete dissolution of the reference material. Furthermore and importantly, the error in the final concentration of the reference translates to an error in the determined concentration of the phosphatidylalkanol, such as e.g. PEth, in the analyzed sample.
Hence, improved compositions comprising phosphatidylalkanols, such as e.g. an improved reference solution of a phosphatidylalkanol, are desired for a simplified, more precise and/or more accurate determination of phosphatidylalkanols, such as e.g. PEth, in samples, such as e.g. human blood samples. In addition, improved regio- and stereo-selective methods of production of such novel and/or presently known PEth-homologues and/or derivatives are also needed.